Acinetobacter baumannii Carbapenemase Producers in Morocco: Genetic Diversity

Introduction: Carbapenem resistance in Acinetobacter baumannii (A. baumannii) is a public health problem worldwide. Although carbapenem resistance is emerging in Morocco, few studies have shown the epidemiological profile of carbapenemase genes in Moroccan healthcare facilities. The aim of this study was to characterize the molecular profile of the carbapenemase enzyme in Acinetobacter baumannii from clinical isolates. Methods: Clinical strains isolated in the laboratory from various samples were subjected to several phenotypic tests. Antibiotic susceptibility and identification were tested using Phoenix 100 (Becton Dickinson Co., Sparks, MD, USA) and Api 20 (bioMérieux, Marcy-l'Etoile, France). Simple phenotypic assays were used to detect carbapenemase oxacillinase (OXA) and metallo-β-lactamase (MBL) production, including the modified Hodge test (MHT) and ethylenediaminetetraacetic acid (EDTA) test. The detection of carbapenemase genes was performed by multiplex and simple polymerase chain reaction (PCR). Results: A total of 140 strains or 100% of isolates contained OXA-51 and ISbA1 sequences, 89% contained OXA-23 and OXA-58 sequences, and 1% contained OXA-24 sequence. The MBL genes were predominated by Verona integron-encoded metallo-β-lactamase (VIM) (56%), followed by Seoul imipenemase (SIM) (39%), German imipenemase (GIM) (37%), São Paulo metallo-β-lactamase (SPM) (13%), imipenemase (IMP) (11%), and New Delhi metallo-β-lactamase (NDM) (4%). Guyana extended-spectrum β-lactamase (GES) was not found in any isolation. Conclusion: Our study shows a high frequency of carbapenem resistance in Acinetobacter baumannii, as it reports a high molecular diversity of carbapenemase-encoding genes, mainly dominated by the carbapenemase ISaba1/OXA-23, which represents an emerging threat in our hospital.


Introduction
Acinetobacter baumannii (A. baumannii) is a gram-negative coccobacillus, opportunistic pathogen that has recently spread worldwide causing epidemics of nosocomial infections, mainly due to its remarkable resistance to antibiotics. Long-term survival in the environment associated with the emergence of drug resistance increases the likelihood of nosocomial transmission. Immunocompromised patients are often the target of this pathogen [1]. OXA-24 [1].
MBLs are enzymes with carbapenemase activity that threaten clinicians, on the one hand, because of their ability to efficiently hydrolyze carbapenems and, on the other hand, because of the high degree of resistance of the bacteria that produce them. Various types of MBLs have been reported worldwide in strains of A. baumannii, including imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), New Delhi metallo-β-lactamase (NDM), São Paulo metallo-β-lactamase (SPM), Seoul imipenemase (SIM), and German imipenemase (GIM) [6].
The detection of different carbapenemases is important to determine the severity of the problem and to guide the application of antibiotic stewardship guidelines to limit the development of other carbapenemresistant variants in the A. baumannii community.
Numerous studies on carbapenem resistance in A. baumannii have been reported worldwide, mainly concerning carbapenemase production. In North Africa, more specifically in Morocco, the presence of the carbapenemase gene in A. baumannii has been detected. Except for sporadic studies [7,8], we are not aware of any other studies on the characterization of carbapenemase genes in A. baumannii in Morocco.
In this regard, the aim of this study is to characterize the molecular profile of carbapenem-resistant A. baumannii among clinical isolates in Morocco at Centre Hospitalier Universitaire (CHU) Hassan II in Fez.

Materials And Methods
This cross-sectional study was conducted at the bacteriology laboratory of Centre Hospitalier Universitaire (CHU) Hassan II in Fez, Morocco, between November 2018 and July 2019. Ethical approval has been granted by the ethical committee of CHU Hassan II University Hospital, Fez. Inpatient specimens collected included blood, catheters, urine, endotracheal secretions from sputum, wound swabs, and cerebrospinal fluid (CSF).

Culture and identification
Samples were inoculated on blood agar, chocolate agar, and MacConkey agar and incubated for 24 hours at 37°C. The identification was based on phenotypic methods, Api 20 gallery (bioMérieux, Marcyl'Etoile, France), and Phoenix 100 gallery (Becton Dickinson Co., Sparks, MD, USA). Polymerase chain reaction (PCR) confirmed the identification of the OXA-51 gene specific to A. baumannii.

Modified Hodge Test (MHT)
The modified Hodge test (MHT) was carried out according to the method described by Lee et al. [9]. Onetenth of the 0.5 McFarland suspension of carbapenem-susceptible Escherichia coli (E. coli) was aseptically buffered on sterile Müller-Hinton agar. An imipenem disc (10 µg) was placed in the center of the plate. A straight line was drawn from the inside to the outside of the plate; each stump has been streaked. A Klebsiella pneumoniae strain was used as a positive control. The dishes were then incubated for 18-24 hours at 37°C and then inspected to detect a space similar to a clover leaf in the inhibition zone of the carbapenem layer at the crossing point of the tested strain and E. coli.

Double-Disc Synergy Test (DDST)
DDST was carried out according to the method described by Lee et al. [9] to detect MBL enzymes. A young culture was diluted to 10 5 CFU/mL and seeded on Müller-Hinton agar with a cotton swab. Two IMP (oxoid) discs were placed on the surface of the agar at 4-5 cm from each other (center to center). Then, 10 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added to one of the two discs. The dish was incubated for 18-24 hours. The presence of a synergistic inhibitory zone was considered positive for MBL.

E-Test MBL
E-test MBL strips (bioMérieux, Solna, Sweden) containing one side of imipenem (4-256 µg/mL) and imipenem (1-64 µg/mL) combined with a fixed concentration of EDTA were used to detect MBL. It is rated according to the manufacturer's instructions. Minimum inhibitory concentration (MIC) levels ≥ 8 for both imipenem and imipenem responses to EDTA are indicative of MBL production.

Total DNA Extraction
Total DNA extraction was carried out using the thermal shock method. A suspension of the strains is studied in 500 µL of distilled water. After boiling the suspension for 10 minutes, it is immediately placed on the ice for two minutes and then centrifuged for 10 minutes at 14,000 rpm. We recovered 300 µL of supernatant in a new 1.5 mL Eppendorf tube and stored it at -20°C.

Gene Amplification by PCR
PCR amplification was carried out using specific primers ( Table 1). The course of the amplification was started by preparing a PCR mix in a final volume of 50 µL. The PCR mix contains 2 µL of DNA, 5 µL of 10× PCR buffer, 2.5 mmol/µL of MgCl2, 100 µm of deoxynucleotide triphosphates (DNTP), 0.4 µL of each primer, and DreamTaq DNA polymerase. The gene amplification program of the oxacillinases OXA-23, OXA-24/40, OXA-51, and OXA-58 and the ISbA1 insertion sequence starts at 94°C for five minutes, followed by 30 cycles. Each cycle consisted of 94°C for 25 seconds, 52°C for 40 seconds, and 72°C for 50 seconds, with a final extension step (72°C for six minutes) to achieve the amplification.

Sequence Analysis
The PCR products positive for the MBL genes, in particular VIM, NDM, and SPM, were purified with the BigDye kit and sequenced. The analysis of the nucleotide sequence was carried out by Basic Local Alignment Search Tool (BLAST) at National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/BLAST).

Discussion
Our study showed a predominance of protected distal specimens (PDS) at 46%, followed by blood cultures and catheters at 18.7%. There is a high prevalence of A. baumannii strains in bronchopulmonary samples, according to several studies [11]. According to many studies, mechanical ventilation is associated with a high incidence of A. baumannii pneumonia, which results in prolonged stays in intensive care units and rapid antibiotic resistance, with a high mortality rate of 45.6%-84.3% [11,12]. The predominance of these infections has been confirmed in many studies [13,14]. Various risk factors associated with Acinetobacter infection in these services may explain this, including immunocompromised patients, longer stays, invasive devices, and the use of broad-spectrum antibiotics. This bacterium can be contaminated frequently and transmitted from healthcare workers to the environment through reservoirs and hands [12].
In our study, the frequency of infection with carbapenem-resistant A. baumannii was higher (93%) in our hospital. This rate in the intensive care unit is high compared to other units. This confirms that A. baumannii is an emerging germ in intensive care [7].
A similar high rate of carbapenem resistance was observed in Morocco (100%) recently by Uwingabiye et al. [15]. It is possible that this high rate is linked to the overuse of carbapenems and third-generation cephalosporins. The selection of strains of A. baumannii resistant to carbapenems has been linked to the pressure exerted by third-generation cephalosporins [16].
Carbapenem resistance can be traced back to several mechanisms. However, the production of MBL and oxacillinase remains the most common mechanism in A. baumannii [1].
Among A. baumannii carbapenemases, oxacillinases are by far the most common [17]. Furthermore, in our isolates, OXA-51, OXA-23, and ISbA1 coexist, explaining their resistance to all antibiotics, including carbapenems. As a matter of fact, the molecular characterization of isolates resistant to carbapenems reveals that in all isolates, in addition to the intrinsic gene OXA-51, the ISbA1 gene is required to overexpress carbapenemase production [18].
We found the coexistence of OXA-51 and OXA-23 genes in 83% of clinical isolates, which is consistent with other studies [19]. As the OXA-23 gene is plasmid-encoded, it may be due to the horizontal spread of plasmid-bearing plasmids that OXA-23 is commonly found in our hospital and in other Moroccan regions [7].
MBLs can hydrolyze all β-lactams except monobactam (e.g., aztreonam). EDTA inhibits their activity, but clavulanic acid does not [20]. We found a diversity of genes responsible for encoding MBLs for these enzymes, thus explaining the high resistance rates of our isolates due to MBL enzymes. Indeed, we detected the gene coding for the VIM in 56% of strains, a higher rate than in other studies at 5.7% [20] and 7% [21]. However, this is slightly consistent with that reported by PeshattiWar et al., the rate being noted at 62.5% [22]. Moreover, we detected the gene coding for SIM in 39% and the gene encoding for GIM in 37%. These three enzymes predominate MBLs in this study.
The SPM gene was detected in 13% of the isolates. Knowing that, the primary report of metallo-β-lactamase from São Paulo was reported in Brazil in an isolate of Pseudomonas aeruginosa (P. aeruginosa), then this enzyme spread throughout Brazil [23], and its first report in Morocco was made by a study already done by our team and published [24].
The IMP gene was found in 11% of the strains. This is a low rate compared to other studies that reported a rate of 95% [25].
The IMP and VIM genes are found along with other resistance genes on integrons associated with transposons. This facilitates translocation and rapid horizontal spread between chromosomes and plasmids [25].
With regard to the NDM-type enzyme, strains of A. baumannii hosting these enzymes are highly observed all over the world, particularly in eastern countries [26]. They have been detected in North Africa, in Algeria [27] and Morocco [15]. The NDM gene was found on several plasmids [28]. The location of NDM on the plasmid facilitates rapid horizontal spread of gram-negative bacteria by conjugation [28]. NDM-1 rates in our study are similar to those in Egypt and Ghana at 8% and 19%, respectively [29].
Due to a lack of resources, this study is not able to sequence many strains nor do a clonality study using pulsed-field gel electrophoresis (PFGE) to determine if our extremely resistant strains are kin.

Conclusions
Our hospital is experiencing an alarming amount of carbapenem resistance in strains of A. baumannii. The high genomic plasticity of this bacterium and its horizontal and clonal design can be explained by the genetic diversity observed in our strains. The control of nosocomial carbapenem-resistant A. baumannii infection and the prevention of dissemination of resistance genes between endemic nosocomial pathogens require local molecular surveillance.

Additional Information Disclosures
Human subjects: All authors have confirmed that this study did not involve human participants or tissue. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.